CONCEPTS IN VIRUS TITRATION

my free school tannzania March 24, 2015
CELL AND TISSUE CULTURE TECHNIQUES
Topic: CONCEPTS IN VIRUS TITRATION
Microtitration of Poliovirus using Vero cell line:
Most of the commonly encountered human viruses produce characteristic cytopathic effect in one or the other cell lines routinely used in virology laboratories. The infectivity titres of these viruses can conveniently be determined by infecting a particular cell line with increasing dilutions of the virus material and determining the highest dilution producing cytopathic effect in 50% of the inoculated cells. The 50% end point dilution which in this case is expressed as TCID 50/ml can be calculated by using either Reed-Muench or Karber formulae. As an example, titration of polio virus I is illustrated in this section.
Materials:
1) Microbiological safety cabinet
2) Sterile test tubes or Penicillin vials
3) Micro pippettes, 200 & 1000 microns
4) CO2 incubator.
5) 96 well flat bottom tissue culture plates (Nunc)
6) Minimum Essential Medium (MEM, Sigma, HI) supplemented with antibiotics
7) Fetal calf serum (FCS)
8) Trypsin versene glucose (TVG)
9) VERO cells
10) Fluid containing PI
11) Inverted microscope
Procedure:
1. Make 10 fold serial dilutions, say from 10-3 to 10-7 of the virus material in MEM containing 2% FCS, dispensed in either test tubes or penicillin vials, changing pipettes or tips with each dilution. Keep the tubes in rack immersed in ice.
2. Transfer 100 ul of each dilution to 4 wells of the micro-titre plate, starting from highest dilution to the lowest.
3. Trypsinise one MD bottle containing a confluent monolayer of Vero cells and count the cells and dilute to 4x 105 cells per ml. In MEM containing10%FCS. Dispense 100 ul of cell suspension in to each of the well containing virus dilution and also include 4 wells as cell control in which 100Ul of cell suspension is mixed with 100 UL of MEM with 2% FCS. While dispensing the virus dilutions and cell suspension it is necessary to keep the plate on ice tray.
4. Cover the plate and keep it in CO2 incubator and adjust the temperature to 37 C.
5. Read the plate under an inverted microscope after 3 to 4 days when a confluent monolayer of Vero cells can be seen in control wells. Look for the cytopathic effect in the wells inoculated with virus dilutions. This consists of rounding of cells, i.e. if 2 of 4 wells inoculated with 10-8 dilution shows cytopathic effect then the titre is 10-8 per 0.1 ml. The titre can also be calculated by the method of Reed and Muench.

A: Calculation of 50% endpoints:
In any biological quantitation, the most desirable endpoint is one representing a situation in which half of the inoculated animals or cells show the reaction (death in the case of animals and in CPE case of cells) and the other half do not. In other words, the endpoint is taken as the highest dilution of the biological material , which produces desired reaction in 50% of the animals or cells. The 50% endpoint can be based on several types of reactions. The most widely used endpoint, based on mortality, is the LD50 (50% lethal dose). This terminology can also be applied to other host systems-for example, tissue cultures - in which the TCID50 represents the dose that gives rise to cytopathic effect in 50% of inoculated cultures. When computing, if closely-placed dilutions are used and in each dilution large number of animals or cells are used, it may be possible to interpolate a correct 50% end point dilution, but it is neither practical nor economical. Reed and Muench devised a simple method for estimation of 50% endpoints based on the large total number of animals, which gives the effect of using at the 2 critical dilutions between which the endpoint lies, larger groups of animals were actually included in these dilutions.